Download In-Fusion™ Advantage PCR Cloning Kit User Manual

User Manual
In-Fusion™ Advantage
PCR Cloning Kit
User Manual
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+1.650.919.7300
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Clontech Laboratories, Inc.
A Takara Bio Company
1290 Terra Bella Ave.
Mountain View, CA 94043
Technical Support (US)
E-mail: [email protected]
www.clontech.com
Cat. Nos. 639616, 639617,
639618, 639619, 639620,
639621, 639622, 639623
& 639624
PT4065-1 (PR9Z3431)
Published January 2010
In-Fusion™ Advantage PCR Cloning Kit User Manual
Table of Contents
I. Introduction.............................................................................................................................. 3
II. In-Fusion Advantage Protocol Overview................................................................................ 4
III. List of Components................................................................................................................. 6
IV. Additional Materials Required................................................................................................ 7
V. PCR and Experimental Preparation........................................................................................ 8
A.Preparation of Linearized Vector by Restriction Digestion............................................................... 8
B. PCR Primer Design.............................................................................................................................. 8
C. PCR Amplification of Insert................................................................................................................11
D. Control Reactions...............................................................................................................................11
VI. Which Protocol Should You Follow?...................................................................................... 12
VII.Protocol I: In-Fusion Cloning Procedure w/Cloning Enhancer Treatment ........................ 12
A.Procedure for Treating Unpurified PCR Inserts with Cloning Enhancer ....................................... 12
B. In-Fusion Cloning Procedure for Cloning Enhancer-Treated PCR Inserts...................................... 12
VIII.Protocol II: In-Fusion Cloning Procedure w/Spin-Column Purification............................. 14
A.Procedure for Spin-Column Purification of PCR Inserts................................................................. 14
B. In-Fusion Cloning Procedure for Spin Column-Purified PCR Inserts............................................. 14
IX. Transformation Procedure..................................................................................................... 15
X. Expected Results.................................................................................................................... 15
XI. Troubleshooting Guide.......................................................................................................... 16
List of Figures
Figure 1. The In-Fusion Cloning Method.........................................................................................................3
Figure 2. In-Fusion Advantage Protocol Flowchart........................................................................................5
Figure 3. Universal primer design for the In-Fusion System........................................................................9
Figure 4. Examples of primers designed for In-Fusion cloning..................................................................10
List of Tables
Table I. In-Fusion Advantage Protocol Outline...............................................................................................4
Table II. Recommended Amount of Vector per In-Fusion Reaction............................................................12
Table III. Recommended Amount of Cloning Enhancer-Treated Insert per In-Fusion Reaction................13
Table IV. Recommended In-Fusion Reactions for Purified Inserts............................................................. 14
Table V. Troubleshooting Guide for In-Fusion Experiments.........................................................................16
Contact Us For Assistance
Customer Service/Ordering:
Technical Support:
Telephone: 800.662.2566 (toll-free)
Telephone: 800.662.2566 (toll-free)
Fax: 800.424.1350 (toll-free)
Fax: 650.424.1064
Web: www.clontech.com
Web: www.clontech.com
E-mail: [email protected]
E-mail: [email protected]
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In-Fusion™ Advantage PCR Cloning Kit User Manual
I. Introduction
In-Fusion™ Advantage PCR Cloning Kits are designed to join multiple pieces of DNA that have
15 bases of homology at their linear ends. A typical use for this technology would be to clone
PCR products into vectors, without the use of restriction enzymes, ligase or phosphatase. InFusion cloning kits, which contain our proprietary In-Fusion Enzyme, let you rapidly generate
very precise constructs. In-Fusion is high-throughput-compatible and universal—it works with
any insert and any vector.
30 min single-tube reaction
x
x
Recombinant
vector
Amplify your
gene of interest
PCR product
Design gene-specific primers
with 15 bp extensions
homologous to vector ends
Any
linearized
vector
The In-Fusion Enzyme
creates single-stranded
regions at the ends of the
vector and PCR product,
which are then fused
due to the 15 bp homology
Figure 1. The In-Fusion Cloning Method.
The In-Fusion Advantage PCR Cloning Method
The In-Fusion method is simple and efficient. First, PCR primers are designed that share 15 bases
of homology with the sequence at the ends of the linearized cloning vector (i.e., at the desired site
of insertion; refer to Section V of this manual). These primers are then used to PCR amplify the
insert DNA. The resulting PCR product is treated with our proprietary Cloning Enhancer or spincolumn purified, and combined with the linearized vector in the In-Fusion cloning reaction.
In general, the In-Fusion reaction consists of a simple 30 minute incubation of the PCR product
with the linearized cloning vector, followed by transformation into E. coli (Figure 1). Each reaction
generates precise constructs with correctly oriented inserts and no additional nucleotides. The
procedure is quite simple, so it is easily automated. With many vectors, such as our pDNR-Dual
(and the linearized pUC19 control vector provided with this Kit), blue/white selection on X-Gal
plates can be used to screen out rare non-linearized vector background.
Although the highest cloning efficiency is achieved with a high-quality PCR product that appears
on an agarose gel as a single, dense band of DNA (with minimal background), PCR products that
contain additional non-specific background can also be cloned using In-Fusion. In such cases,
the target PCR product is not treated with the Cloning Enhancer. It is, instead, isolated by gel
extraction and spin column-purified before cloning. Figure 2 illustrates the differences in the
experimental workflow required for Cloning Enhancer treated PCR products versus those that
are spin column-purified.
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In-Fusion™ Advantage PCR Cloning Kit User Manual
II. In-Fusion Advantage Protocol Overview
The table below is a general outline of the protocol used in the In-Fusion Advantage PCR Cloning Kits. This outline
is further illustrated in Figure 2. Please refer to the specified pages for details on performing each step.
Step
Table I. In-Fusion Advantage Protocol Outline
Action
1
Select a base vector and identify the insertion site. Linearize the
vector by restriction enzyme digestion or inverse PCR and purify.
2
Design PCR primers for your gene of interest with 15 bp extensions
(5’) that are homologous to the ends of the linearized vector.
8-10
3
Amplify your gene of interest with a high-fidelity DNA polymerase.
11
4
Verify on an agarose gel that your target DNA has been amplified
and determine the integrity of the PCR product. If a single prominent band of desired size is obtained, you can EITHER treat your
insert with Cloning Enhancer (follow Protocol I), OR treat your
insert with DpnI and spin-column purify (follow Protocol II). If a
non-specific background smear or multiple bands are visible on
your gel, isolate your target fragment by gel extraction and spincolumn purify (follow Protocol II).
11-12
5
Treat your target fragment with Cloning Enhancer OR spin-column
purify.
Cloning Enhancer Protocol I (p.12)
OR
Spin-Column Protocol II (p.14)
6
Determine the appropriate amount of Cloning Enhancer-Treated or Cloning Enhancer Protocol I (p.12)
Spin Column-Purified PCR product (insert) and vector to use in your
OR
In-Fusion cloning reaction.
Spin-Column Protocol II (p.14)
7
Set up your In-Fusion cloning reaction:
2 μl of 5X In-Fusion Reaction Buffer
1 μl of In-Fusion Enzyme
X μl of Vector
X μl of Insert
X μl of dH20 to a Total Reaction Volume of 10 μl. Mix well.
Cloning Enhancer Protocol I (p.13)
OR
Spin-Column Protocol II (p.14)
8
Incubate the reaction for 15 min at 37°C, followed by 15 min at
50°C, then place on ice.
Cloning Enhancer Protocol I (p.13)
OR
Spin-Column Protocol II (p.14)
9
Bring the reaction volume up to 50 µl with TE buffer (pH 8), and
mix well.
Cloning Enhancer Protocol I (p.13)
OR
Spin-Column Protocol II (p.14)
10
Transform competent cells with 2.5 μl of the diluted reaction
mixture from Step 9.
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15
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In-Fusion™ Advantage PCR Cloning Kit User Manual
II. In-Fusion Advantage Protocol Overview, continued
Generate a linearized vector
Step 1
Design gene-specific primers with 15 bp extensions homologous to vector ends
Step 2
15 bp
M 1
2
Amplify your gene of interest
M
1
2
Step 3
15 bp
PCR product
If you obtain pure PCR product
with a single specific band,
no background
If you obtain PCR product
with non-specific background,
isolate the target fragment
by gel extraction
Step 4
Treat with DpnI
Spin-Column purify
OR
Step 5
Step 5
Spin-Column Protocol II
Cloning Enhancer Protocol I
Treat with Cloning Enhancer
Add 2 µl of Cloning Enhancer to 5 µl of PCR product
and incubate 15 min at 37º C, 15 min at 80º C
Step 5.2
Determine the volume of Cloning Enhancertreated insert and linearized vector to use
in the In-Fusion reaction
Mix the purified PCR insert and vector
together at a 2:1 molar ratio
Step 6
Step 6
Set up the In-Fusion cloning reaction
Step 7
2 μl 5X Buffer
1 μl In-Fusion Enzyme
x μl Vector
x μl Insert
x μl dH2O
15 min at 37º C
15 min at 50º C
10 μl Total Volume
Incubate cloning reaction
Step 8
x
x
Recombinant
vector
Dilute reaction with TE Buffer
Step 9
Transform competent E. coli with the diluted reaction mixture
Step 10
Screen clones
Figure 2. In-Fusion Advantage Protocol Flowchart
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In-Fusion™ Advantage PCR Cloning Kit User Manual
III. List of Components
The In-Fusion Advantage PCR Cloning Kits are available in 10, 50 and 100 reaction sizes, with or without
Cloning Enhancer or NucleoSpin® Extract II.
Store NucleoSpin Extract II at room temperature.
Store all other components at –20°C.
In-Fusion™ Advantage PCR Cloning Kits
Components
Cat. Nos.
639619
639620
639621
Rxns.
10 rxns
50 rxns
100 rxns
10 μl
50 μl
100 μl
20 μl
100 μl
200 μl
5 μl
5 μl
5 μl
10 μl
10 μl
10 μl
In-Fusion Enzyme
5X In-Fusion Reaction Buffer
pUC19 Control Vector,*
linearized (50 ng/μl)
Component
Amounts
2 kb Control Insert (40 ng/μl)
In-Fusion™ Advantage PCR Cloning Kits w/NucleoSpin®
Components
Cat. Nos.
639622
639623
639624
Rxns.
10 rxns
50 rxns
100 rxns
In-Fusion Enzyme
10 μl
50 μl
100 μl
5X In-Fusion Reaction Buffer
20 μl
100 μl
200 μl
5 μl
5 μl
5 μl
2 kb Control Insert (40 ng/μl)
10 μl
10 μl
10 μl
NucleoSpin Extract II
10 preps
50 preps
100 preps
pUC19 Control Vector,*
linearized (50 ng/μl)
Component
Amounts
In-Fusion™ Advantage PCR Cloning Kits w/Cloning Enhancer
Components
Cat. Nos.
639616
639617
639618
Rxns.
10 rxns
50 rxns
100 rxns
In-Fusion Enzyme
10 μl
50 μl
100 μl
5X In-Fusion Reaction Buffer
20 μl
100 μl
200 μl
5 μl
5 μl
5 μl
2 kb Control Insert (40 ng/μl)
10 μl
10 μl
10 μl
Cloning Enhancer
50 μl
100 μl
200 μl
pUC19 Control Vector,*
linearized (50 ng/μl)
Component
Amounts
*These Kits contain only enough vector for the control reactions.
pUC19 Control Vector Information is available on line at www.clontech.com/support/vectors.asp
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In-Fusion™ Advantage PCR Cloning Kit User Manual
IV. Additional Materials Required
The following materials are required but not supplied:
•
TE Buffer (pH 8.0) required for diluting the In-Fusion reaction prior to transformation
10 mM Tris-HCl
1 mM EDTA
•
Sodium Acetate (3 M) required only if concentrating DNA by precipitation
•
Glycogen (20 µg/µl) required only if concentrating DNA by precipitation
•
Ampicillin (100 mg/ml stock) or other antibiotic required for plating the In-Fusion reaction
•
LB (Luria-Bertani) medium (pH 7.0)
•
LB/antibiotic plates
•
SOC medium
2%
0.5%
10 mM
2.5mM
10 mM
20 mM
Tryptone
Yeast Extract
NaCl
KCl
MgCl2•6H2O
glucose
1. For 1 liter, dissolve 20 g of tryptone, 5 g of yeast extract, and 0.5 g of NaCl in 950 ml of deionized
H2O.
2. Prepare a 250 mM KCl solution by dissolving 1.86 g of KCl in deionized H2O for a total volume
of 100 ml. Add 10 ml of this stock KCl solution to the solution prepared in Step 1.
3. Adjust pH to 7.0 with 5 M NaOH, then bring the volume to 980 ml with deionized H2O.
4. Prepare a 1 M solution of MgCl2 by dissolving 20.33 g of MgCl2•6H2O in deionized H2O for a
total volume of 100 ml.
5. Autoclave both solutions on liquid cycle at 15 lbs/in2 for 20 min.
6. Meanwhile, make a 2 M solution of glucose by dissolving 36 g of glucose in deionized H2O for a
total volume of 100 ml. Filter-sterilize this solution.
7. Let the autoclaved solutions cool to about 55°C, then add 10 ml of the filter-sterilized 2 M glucose solution and 10 ml of 1 M MgCl2. Store at room temperature or 4°C.
• Competent Cells
We recommend the use of Fusion-Blue™ Competent Cells or any commercially-available competent
cells (e.g., DH10B™, DH5α™) that have a transformation efficiency ≥ 1.0 x 108 cfu/µg. Fusion-Blue
Competent Cells are available in 24-transformation (Cat. No. 636700) and 96-transformation (Cat.
No. 636758) formats.
• Cloning Enhancer (Cat. Nos. 639613, 639614 & 639615) [Optional]
Cloning Enhancer is provided with some of the In-Fusion Advantage PCR Cloning Kits and can also
be purchased separately. Cloning Enhancer removes background template DNA and PCR residue,
eliminating the need for PCR insert purification prior to cloning when a single PCR product (i.e., no
background) is obtained (See Section V.C.).
• Spin Columns—NucleoSpin® Extract II (Cat. Nos. 740609.10, 740609.50 & 740609.250) [Optional]
Spin columns can be used to purify PCR products, eliminating the need for gel extraction when a
single PCR product (i.e., no background) is obtained (See Section V.C.). However, Spin Columns can
also be used in conjunction with gel purification (e.g., if non-specific background or multiple bands
are visible on an agarose gel). When spin columns are needed, we recommend NucleoSpin® Extract
II. NucleoSpin® Extract II is provided with some of the In-Fusion Advantage PCR Cloning Kits and
can also be purchased separately.
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In-Fusion™ Advantage PCR Cloning Kit User Manual
V. PCR and Experimental Preparation
PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.
A. Preparation of Linearized Vector by Restriction Digestion
To achieve a successful In-Fusion reaction, you must first generate a linearized vector (with a very low background of uncut vector present). The linearized vector can be generated using restriction enzymes (single
or double digests) or by PCR.
Due to differences in cutting efficiencies, different restriction enzymes will generate different amounts of
background. Generally speaking, two enzymes cut better than any single enzyme. Efficiency of digestion
will always be better if the restriction sites are as far apart as possible. In addition, increasing the enzyme
digestion time and the digestion reaction volume will reduce the background.
Prepare a linearized vector by restriction enzyme digestion as follows.
1. We recommend cutting the vector with two different enzymes to reduce background, unless there is
only one site available for cloning.
2. Incubate your restriction digest as directed by the restriction enzyme supplier. For many enzymes,
incubation from 3 hours to overnight can increase linearization and reduce background.
3. After digestion, purify the linearized vector using any available PCR purification kit. We recommend
using the NucleoSpin® Extract II Kit.
4. [Control] Check the background of your vector by transforming 5–10 ng of the linearized and
purified vector into competent cells (See Transformation Procedure, Section IX).
If the background is high, continue digesting the vector for a longer time after the addition of more restriction enzyme(s). Incubate 2 hours to overnight. Gel purify the remainder of the vector and transform
again.
B.PCR Primer Design
Primer design and quality are critical for the success of the In-Fusion reaction. In-Fusion allows you to join
two or more fragments, e.g. vector and insert (or multiple inserts), as long as they share 15 bases of homology at each end. Therefore, In-Fusion PCR primers must be designed in such a way that they generate PCR
products containing ends that are homologous to those of the vector. Figure 3 outlines the guidelines for
primer design and Figure 4 gives specific examples of In-Fusion PCR primers.
When designing In-Fusion PCR primers, consider the following:
1. Every In-Fusion primer must have two characteristics: The 5’ end of the primer must contain 15 bases
that are homologous to 15 bases at one end of the DNA fragment to which it will be joined (i.e., the vector
or another insert). The 3’ end of the primer must contain sequence that is specific to the target gene.
2. The 3’ portion of each primer should:
•
be gene-specific.
•
be between 18–25 bases in length, and have a GC-content between 40–60%.
•
have a melting temperature (Tm) between 58–65°C. The Tm difference between the forward and
reverse primers should be ≤ 4°C, or you will not get good amplification. Note: The Tm should be
calculated based upon the 3’ (gene-specific) end of the primer, and NOT the entire primer. If the
calculated Tm is too low, increase the length of the gene-specific portion of the primer until you
reach a Tm of between 58–65°C.
•
not contain identical runs of nucleotides.The last five nucleotides at the 3’ end of each primer should
contain no more than two guanines (G) or cytosines (C).
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V. PCR and Experimental Preparation, continued
3. Avoid complementarity within each primer to prevent hairpin structures, and between primer pairs to
avoid primer dimers.
4. You can perform a BLAST search to determine if the 3’ portion of each primer is unique and specific (at
www.ncbi.nlm.nih.gov/BLAST/).
5. Clontech provides an online tool (at http://bioinfo.clontech.com/infusion/) that simplifies In-Fusion
PCR primer design for standard cloning reactions. Simply provide your vector sequence, the restriction
enzyme(s) used to linearize the vector (if that is the chosen method for linearization), and the primer
sequence required to amplify your region of interest.
6. We generally use desalted oligonucleotide primers in PCR reactions. However, primer quality can depend on the vendor and varies from lot to lot. If your primer quality is particularly poor (i.e., has many
premature termination products), or your primers are longer than 45 nucleotides, they may need to be
PAGE purified; however, we usually find this is unnecessary.
Forward Primer
NNNNNNNNNNNNNNN
Linearized
Vectors with
5' Overhangs
NNNNNNNNNNNNNN
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
append with your specific sequence
*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
*
NNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
Reverse Primer
Forward Primer
NNNNNNNNNNNNNNN
Linearized
Vectors with
Blunt ends
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
NNNNNNNNNNNNNNNNNN
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
*
*
NNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
Reverse Primer
Forward Primer
NNNNNNNNNNNNNNN
Linearized
Vectors with
3' Overhangs
NNNNNNNNNNNNNNNNNNNNNN
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
*
NNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
Reverse Primer
Guidelines for universal primer design
To determine the 15 b homology sequence to be incorporated into each
primer, start at the 5’ end of each DNA strand in the linearized vector (*).
The region of homology for a particular primer consists of bases that are
complementary to the first 15 bases at the 5’ end of a particular DNA strand.
This means that the bases complementary to 5' overhangs are included in
the primer sequence, but the bases in 3’ overhangs are not.
Brackets indicate bases
to be included in the
15 b region of homology
Figure 3. Universal primer design for the In-Fusion System. Successful insertion of a PCR fragment requires that the
PCR insert share 15 bases of homology with the ends of the linearized vector. This sequence homology is added to the
insert through the PCR primers. For vectors with sticky ends, bases complementary to 5’ overhangs are included in the
primer sequence; bases in the 3’ overhangs are not. See Figure 4 for specific examples. An online tool is also provided
to assist in primer design and can be found at http://bioinfo.clontech.com/infusion/.
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V. PCR and Experimental Preparation, continued
5' Forward Primer
SalI
5'-G AAG TTA TCA GTC GAC XXX XX...-3'
A
5'-...ATA CAT TAT ACG AAG TTA TCA G
3'-...TAT GTA ATA TGC TTC AAT AGT CAG CT
pDNR-CMV sequence
AGC TTT CTA GAC CAT TCG TTT GGC G...-3'
AA GAT CTG GTA AGC AAA CCG C...-5'
3'-...X XXX XXT TCG AAA GAT CTG GTA-5'
HindIII
3' Reverse Primer
SmaI
5'-ACC GGA CAT ATG CCC GGG XXX...-3'
B
5'-...TCA GTC GAC GGT ACC GGA CAT ATG CCC
3'-...AGT CAG CTG CCA TGG CCT GTA TAC GGG
GGG AAT TCC TGC AGG ATC CGC T...-3'
CCC TTA AGG ACG TCC TAG GCG A...-5'
3'-...XXX GGG CCC TTA AGG ACG TCC-5'
SmaI
KpnI
5'-AG TTA TCA GTC GAC GGT ACC XXX...-3'
C
5'-...CAT TAT ACG AAG TTA TCA GTC GAC GGT AC
3'-...GTA ATA TGC TTC AAT AGT CAG CTG C
C GGA CAT ATG CCC GGG AAT T...-3'
CA TGG CCT GTA TAC GGG CCC TTA A...-5'
3'-...XXX CCA TGG CCT GTA TAC GGG CC-5'
KpnI
Figure 4. Examples of primers designed for In-Fusion cloning. The above figure shows examples of primers designed with
recognition sites for restriction enzymes that generate: 5’ overhangs (Panel A), blunt ends (Panel B), and 3’ overhangs
(Panel C). The primer sequences are shown in bold. The Xs represent bases corresponding to the gene or sequence of
interest. Additional nucleotides (indicated with a black box) have been added to each primer in order to reconstruct the
restriction sites. They are not part of the 15 bases of sequence homology.
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V. PCR and Experimental Preparation, continued
C. PCR Amplification of Insert
For most DNA polymerases, 10–100 ng of plasmid DNA is typically enough to use as a PCR template. However, if you are amplifying from a pool of cDNA, the amount of template DNA required depends on the
relative abundance of the target message in your mRNA population.
The In-Fusion method is not affected by the presence or absence of A-overhangs, so you can use any
thermostable DNA polymerase for amplification, including proofreading enzymes. For the best results, we
recommend using our Advantage® HD Polymerase Mix (Cat. No. 639241), which offers high-fidelity, efficient
amplification of long gene segments (>1 kb), and automatic hot start for increased specificity and reduced
background. For high yields and error-free amplification of inserts up to 5 kb, we recommend using the
Advantage® HF 2 enzyme supplied in our Advantage HF 2 PCR Kits (Cat. Nos. 639123 & 639124).
If you will be performing PCR with Advantage HD Polymerase, we recommend using the following amounts
of template (for a 50 µl reaction):
Human Genomic DNA
E. coli Genomic DNA
λ DNA
Plasmid DNA
5 ng–200 ng
100 pg–100 ng
10 pg–10 ng
10 pg–1 ng
If you choose not to use Advantage HD, we recommend that you use a robust, high fidelity, thermostable
DNA polymerase that is capable of hot start PCR.
When PCR cycling is complete, analyze your PCR product by agarose gel electrophoresis to confirm that you
have obtained a single DNA fragment and to estimate the concentration of your PCR product. Quantify the
amount of DNA by measuring against a known standard or DNA mass ladder ladder run on the same gel.
Attention
IMPORTANT:
Following PCR, verify on an agarose gel that your target fragment has been amplified. If a single band of
desired size is obtained, you can EITHER treat your PCR product with Cloning Enhancer (follow Protocol I)
OR treat your PCR product with DpnI and spin-column purify (follow Protocol II). However, if non-specific
background or multiple bands are visible on your gel, we recommend that you isolate your target fragment by gel extraction, then spin-column purify (follow Protocol II).
D. Control Reactions
When using the In-Fusion kit for the first time, we strongly recommend that you perform the positive and
negative control reactions in parallel with your In-Fusion cloning reaction. Performing the control reactions
will verify that the system is working properly. The 2 kb Control Insert included in the In-Fusion Advantage
PCR Cloning Kits has already been purified, so there is no need for further treatment prior to the cloning
reaction.
To perform the control reactions, proceed with the In-Fusion Cloning Procedure for Spin Column-Purified
PCR Inserts (Section VIII.B).
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VI. Which Protocol Should You Follow?
Following PCR, verify by agarose gel electrophoresis that your target fragment has been amplified. If a single
band of the desired size is obtained, you can EITHER treat your PCR product with Cloning Enhancer (follow
Protocol I), OR treat your PCR product with DpnI and spin-column purify (follow Protocol II). However, if nonspecific background or multiple bands are visible on your gel, we recommend that you isolate your target
fragment by gel extraction, then spin-column purify (follow Protocol II).
VII.Protocol I: In-Fusion Cloning Procedure w/Cloning Enhancer Treatment
A. Procedure for Treating Unpurified PCR Inserts with Cloning Enhancer
Attention
IMPORTANT:
DO NOT treat purified PCR products with the Cloning Enhancer.
Before setting up the In-Fusion cloning reaction, treat unpurified PCR products (e.g. inserts) as follows:
1. Add 2 µl of Cloning Enhancer to 5 µl of the PCR reaction.
Protocol
BREAK
2. Incubate at 37°C for 15 minutes, then at 80°C for 15 minutes in a PCR thermal cycler. If you used more
than 100 ng of DNA as a template in the PCR reaction, extend the 37°C incubation step to 20 minutes.
If you are using a water bath or heat block rather than a thermal cycler, extend each of the incubation
steps to 20–25 minutes.
3. Proceed with the In-Fusion Cloning Procedure for Cloning Enhancer-Treated PCR Inserts (Section VII.B).
If you cannot proceed immediately, store treated PCR reactions at –20°C until you are ready.
B. In-Fusion Cloning Procedure for Cloning Enhancer-Treated PCR Inserts
Attention
IMPORTANT:
Before proceeding to the cloning reaction, be sure your target insert has been pretreated with the Cloning Enhancer,
as described in Section A (above). DO NOT follow this procedure if your insert has been purified.
1. Use Table II to determine the amount of linearized vector to use in your In-Fusion reaction.
Table II. Recommended amount of vector
per in-fusion reaction
Protocol
Vector Size
Recommended Nanograms (ng)
<4 kb
100 ng
4 to 6 kb
100 to 150 ng
6 to 10 kb
200 ng
>10 kb
Up to 400 ng
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VII. Protocol I: In-Fusion Cloning Procedure w/Cloning Enhancer, continued
2. Use Table III to determine the amount of Cloning Enhancer-Treated PCR Insert (from Section VII, Part A)
to use in your In-Fusion reaction.
Table III. Recommended AMOUNT of cloning enhancer-treated
insert per in-fusion reaction*
Protocol
InsertSize
Recommended Microliters (µl)
<1 kb
1 µl
1 to 4 kb
1 to 2 µl
4 to 8 kb
4 µl
8 to 12 kb
7 µl
*If you obtain a low product yield from your PCR reaction, we recommend
adding more of the Cloning Enhancer treated insert (up to 7 µl).
3. Set up the In-Fusion cloning reaction:
5X In-Fusion Reaction Buffer
2 µl
In-Fusion Enzyme
Vector
1 µl
x µl*
Cloning Enhancer-Treated PCR Insert x µl*
dH2O (as needed)
Total Volume
x µl
10 µl
*For reactions with larger volumes of vector and insert (> 7 µl of vector + insert), double the amount of reaction buffer
and enzyme, and add dH20 for a total volume of 20 µl.
4. Adjust the total reaction volume to 10 µl using deionized H2O and mix the reaction.
5. Incubate the reaction for 15 min at 37°C, followed by 15 min at 50°C, then place on ice.
6. Bring the reaction volume up to 50 µl* with TE buffer (pH 8), and mix well.
*For some cell strains, it may be better to dilute the reaction with TE buffer to a volume of 100 μl.
If your cloning efficiency is low, you may obtain better results if you dilute the reaction further.
7.
BREAK
Continue to the Transformation Procedure (Section IX). If you cannot transform cells immediately, store
the cloning reactions at –20°C until you are ready.
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VIII. Protocol II: In-Fusion Cloning Procedure w/Spin-Column Purification
A. Procedure for Spin-Column Purification of PCR Inserts
Protocol
1. If non-specific background bands are observed on an agarose gel, isolate your target fragment by gel
extraction, then spin-column purify. If a single band of the desired size is obtained, add 1 μl of DpnI to
50 μl of the PCR reaction and incubate at 37°C for 60 min, then spin-column purify.
2. Spin-column purify your PCR product (e.g., insert) by using a silica-based purification system, such as
NucleoSpin® Extract II. During purification, avoid nuclease contamination and exposure of the DNA to
UV light for long periods of time.
3. After purification, proceed with the In-Fusion Cloning Procedure for Spin Column-Purified PCR Inserts
(Section VIII.B).
B. In-Fusion Cloning Procedure for Spin-Column Purified PCR Inserts
In general, maximum cloning efficiency is achieved when using a 2:1 molar ratio of insert:vector. Typically,
100 ng of a 4–5 kb linearized vector plus 50 ng of a 1 kb PCR product is found to work well in an In-Fusion
reaction. If the size of your vector or PCR product is different from this, adjust the amount of your input
DNA. Clontech provides an online tool to assist in determining the correct amount of insert and vector to
achieve a 2:1 ratio (http://bioinfo.clontech.com/infusion/).
Table Iv. Recommended in-fusion reactions for purified inserts
Rxn Component
Cloning Rxn
Negative Control
Rxn
Positive Control
Rxn
Purified PCR insert
50–200 ng
–
2 µl**
Linearized vector
100–400 ng
1 µl*
1 µl*
5X In-Fusion Reaction Buffer
2 µl
2 µl
2 µl
In-Fusion Enzyme
1 µl
1 µl
1 µl
Deionized water
to 10 µl
to 10 µl
to 10 µl
*Use 1.0 µl of the linearized pUC19 Control Vector (50 ng/µl) included in the Kit.
**Use 2 µl of the 2 kb Control Insert (40 ng/ml) included in the Kit.
1. Mix your purified PCR insert and vector together in a 2:1 molar ratio.
2. Set up the In-Fusion cloning reaction:
Protocol
5X In-Fusion Reaction Buffer
2 µl
In-Fusion Enzyme
1 µl
Vector
x µl*
Purified PCR Insert
x µl*
dH2O (as needed)
x µl
Total Volume
10 µl
*For reactions with larger volumes of vector and insert (> 7 µl of vector + insert), double the amount of
reaction buffer and enzyme, and add dH20 for a total volume of 20 µl.
3. Adjust the total reaction volume to 10 µl using deionized H2O and mix the reaction
4. Incubate the reaction for 15 min at 37°C, followed by 15 min at 50°C, then place on ice.
5. Bring the reaction volume up to 50 µl* with TE buffer (pH 8) and mix well.
*For some cell strains, it may be better to dilute the reaction to 100 μl with TE buffer If your
cloning efficiency is low, you may obtain better results if you dilute the reaction further.
BREAK
6. Continue to the Transformation Procedure (Section IX). If you cannot transform cells immediately, store
the cloning reactions at –20°C until you are ready.
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IX. Transformation Procedure
Protocol
In addition to the cloning reaction, we recommend that you perform positive and negative control transformations. The positive control should consist of a circular vector of known concentration (competent cells
should give >1 x 108 cfu/µg), and the negative control should consist of a known amount of your linearized
vector (see Section X for Expected Results).
1. Transform competent cells with 2.5 µl of the diluted In-Fusion reaction mixture. Follow the transformation protocol provided by your competent cell manufacturer.
IMPORTANT:
DO NOT add more than 5 µl of the diluted reaction to 50 µl of competent cells. More is not better. Using
too much of the reaction mixture inhibits the transformation. For example, 0.5–1 µl of an undiluted In-Fusion
reaction in 50 µl of cells typically yields over 1,000 colonies, while 2 µl of the same reaction will yield fewer than
100 colonies. Since it can be difficult to pipette 1 µl accurately, it is necessary to dilute the In-Fusion reaction
with TE buffer (see Section VII.B.6 and Section VIII.B.5) before performing the transformation.
Attention
2. Place 1/10th of each transformation reaction (25–50 µl) into separate tubes and bring the volume to 100
µl with SOC medium. Spread each diluted transformation reaction on a separate LB plate containing
an antibiotic appropriate for the cloning vector (i.e., the control vector included with the Kit requires
100 µg/ml of ampicillin).
3. Centrifuge the remainder of each transformation reaction at 6000 rpm for 5 min. Discard the supernatant
and resuspend each pellet in 100 µl fresh SOC medium. Spread each sample on a separate LB plate
containing the appropriate antibiotic. Incubate all of the plates overnight at 37°C.
4. The next day, pick individual isolated colonies from each experimental plate. Isolate plasmid DNA using
a standard method of your choice (e.g. miniprep). To determine the presence of insert, analyze the DNA
by restriction digestion or PCR screening.
X. Expected Results
The positive control plates typically develop several hundred white colonies when using cells with a
minimum transformation efficiency of 1 x 108 cfu/μg. The negative control plates should have few colonies.
The number of colonies on your experimental plates will depend on the amount and purity of the PCR
product and linearized vector used for the In-Fusion cloning reaction.
• The presence of a low number of colonies on both plates—typically, a few dozen colonies—
is indicative of either transformation with too much of the reaction, or poor DNA/primer quality.
• The presence of many (hundreds) of colonies on the negative control is indicative of incomplete
vector linearization.
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XI. Troubleshooting Guide
If you do not obtain the expected results, use the following guide to troubleshoot your experiment. To
confirm that your kit is working properly, perform the control reactions.
Table V. Troubleshooting Guide for In-Fusion EXPERIMENTS
A. No or Few Colonies Obtained from Transformation
Description of
Problem
Low
transformation
efficiency
Explanation
Solution
Transformed with too
much In-Fusion
reaction
Do not add more than 5 µl of the diluted In-Fusion reaction
to 50 µl of competent cells (see Section IX for details).
Suboptimal dilution of
In-Fusion reaction
For some cell strains, it may be better to dilute the In-Fusion
reaction with TE buffer up to a total volume of 100 μl (see
Section VII.B, Step 6 OR Section VIII.B, Step 5).
Suboptimal PCR
product
Repeat PCR amplification and purify product using a
different method of purification. Alternatively, perform
phenol:chloroform extraction on your original PCR
product, followed by ethanol precipitation.
Bacteria were not
competent
Check transformation efficiency. You should obtain >1 x 108
cfu/µg; otherwise use fresh competent cells.
It is imperative to obtain the highest DNA concentration
Low DNA concentration possible in your In-Fusion reaction. Either the amount of vector
in reaction
or the amount of PCR fragment was too low. We recommend
using between 100 ng and 400 ng of vector, depending on
its size (see Table II).
Wrong molar ratio
Low quality DNA
fragments
Gel purification
introduced
contaminants
Primer sequences are
incorrect
Low cloning
Cloning Enhancer may
efficiency in the
not have been
experiment, but
inactivated properly
not in the control
The molar ratio of PCR fragment to linear vector used in
the In-Fusion protocol may not have been optimal. We
recommend using between 100 ng and 400 ng of vector, and
50 to 200 ng of insert. Clontech provides an online tool to
assist in determining the correct amount of insert and vector to
achieve a 2:1 ratio (http://bioinfo.clontech.com/infusion/).
If your insert was gel purified, it is imperative to obtain the
highest DNA concentration possible in your In-Fusion reaction. The total volume of purified vector and insert should
not exceed 5 µl.
When possible, optimize your PCR amplification reactions
such that you generate pure PCR products and use the
Cloning Enhancer (see Section VII.A for details).
Check primer sequences to ensure that they provide 15 bases
of homology with the region flanking the insertion site (see
Section V.B).
Extend the inactivation step of the Cloning Enhancer to
80°C for 20–25 minutes.
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XI. Troubleshooting Guide, continued
Table V. Troubleshooting Guide for In-Fusion EXPERIMENTS
B. Large Numbers of Colonies Contained No Insert
Description of
Problem
Explanation
Solution
Incomplete linearization It is important to remove any uncut vector prior to
use in the In-Fusion reaction. If necessary, recut your
of your vector
vector and gel purify.
Large numbers of
Contamination of
colonies obtained with In-Fusion reaction by
no insert
plasmid with same
antibiotic resistance
Plates too old or
contained incorrect
antibiotic
If your insert was amplified from a plasmid, closed
circular DNA may have carried through purification
and contaminated the cloning reaction:
a) To ensure the removal of any plasmid
contamination, we recommend linearizing the
template DNA before performing PCR.
b) If you spin-column purify your insert, be
sure to treat the PCR product with DpnI
before purification in order to remove contaminating template DNA (see Section VIII.A.1).
Be sure that your antibiotic plates are fresh (<1
month old). Check the antibiotic resistance of your
fragment.
C. Clones Contained Incorrect Insert
Large number of colonies contain incorrect
insert
Your PCR product
contained non-specific
sequences
If your PCR product is not a single distinct band, then
it may be necessary to gel purify the PCR product to
ensure cloning of the correct insert. See Section VI for
more information.
Notice to Purchaser
Clontech products are to be used for research purposes only.They may not be used for any other purpose, including, but not limited to, use in drugs,
in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or
used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc.
This product is covered by U.S. Patent No 7,575,860 and European Patent No. EP1741787.
Fusion-Blue™ Cells are manufactured and tested by Novagen for Clontech Laboratories, Inc.
Novagen® is a registered trademark of EMD Biosciences, Inc.
U.S. Patent No. 5,436,149 for LA Technology is owned by Takara Bio Inc.
TaqStart® Antibody and other Hot Start Antibodies are licensed under U.S. Patent No. 5,338,671.
DH5™, DH5a™, DH10B™ and ElectroMax™are trademarks, and Max Efficiency® is a registered trademark of Invitrogen Corporation.
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Clontech, the Clontech Logo and all other trademarks are the property of Clontech Laboratories, Inc., unless noted otherwise. Clontech is a Takara
Bio Company. ©2010 Clontech Laboratories, Inc.
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